Caspase-1 in Cx3cr1-expressing cells drives an IL-18-dependent T cell response that promotes parasite control during acute T. gondii infection

Inflammasome activation is a robust innate immune mechanism that promotes inflammatory responses through the release of alarmins and leaderless cytokines, including IL-1α, IL-1β, and IL-18. Various stimuli, including infectious agents and cellular stress, cause inflammasomes to assemble and activate caspase-1. Then, caspase-1 cleaves targets that lead to pore formation and leaderless cytokine activation and release. Toxoplasma gondii has been shown to promote inflammasome formation, but the cell types utilizing caspase-1 and the downstream effects on immunological outcomes during acute in vivo infection have not been explored. Here, using knockout mice, we examine the role of caspase-1 responses during acute T. gondii infection globally and in Cx3cr1-positive populations. We provide in vivo evidence that caspase-1 expression is critical for, IL-18 release, optimal interferon-γ (IFN-ψ) production, monocyte and neutrophil recruitment to the site of infection, and parasite control. Specifically, we find that caspase-1 expression in Cx3cr1-positive cells drives IL-18 release, which potentiates CD4+ T cell IFN-γ production and parasite control. Notably, our Cx3cr1-Casp1 knockouts exhibited a selective T cell defect, mirroring the phenotype observed in Il18 knockouts. In further support of this finding, treatment of Cx3cr1-Casp1 knockout mice with recombinant IL-18 restored CD4+ T cell IFN-γ responses and parasite control. Additionally, we show that neutrophil recruitment is dependent on IL-1 receptor accessory protein (IL-1RAP) signaling but is dispensable for parasite control. Overall, these experiments highlight the multifaceted role of caspase-1 in multiple cell populations contributing to specific pathways that collectively contribute to caspase-1 dependent immunity to T. gondii. AUTHOR SUMMARY When a cell undergoes inflammatory cell death, termed pyroptosis, cellular content is released and has the potential to stimulate immune responses. Our work highlights that in the context of T. gondii infection, distinct cell populations undergo pyroptosis each of which has different impacts on how the immune system responds. These findings suggest a collaborative effort of multiple cell types undergoing pyroptosis for optimal immunity to infection. Using a cell-type specific knockout to render macrophages incapable of undergoing pyroptosis, we find that macrophage pyroptosis reinforces adaptive immune cell function, while other population’s pyroptosis stimulates the recruitment of innate immune cells into the infected tissue. We go on to identify a specific molecule, IL-18, is released from macrophage pyroptosis that reinforces adaptive immune cell function. By reintroducing IL-18 into the macrophage knockout mice, we successfully restored adaptive immune cell function thereby facilitating the recovery of parasite control. This study outlines the impact of pyroptosis on immunity to T. gondii and stratifies the effects from separate cell populations and their associated downstream pathways.


INTRODUCTION
Innate pathogen sensing via pattern recognition receptors (PRRs) instructs and mobilizes the immune system in response to infections.The diversity of PRRs and the molecules they recognize tailor the immune response generated.These PRRs include toll-like receptors (TLR), Nod-like receptors (NLR), and C-type lectin receptors (1).Specifically, activation of some NLRs triggers the formation of a multi-protein complex known as the inflammasome.The effector protein of the inflammasome is caspase-1, which cleaves substrates including those that create pores in the cell membrane.Consequently, this process prompts inflammatory cell death and the release of immune-activating cytosolic contents, including leaderless cytokines which do not leave the cell through conventional secretion pathways and rely on proteolytic cleavage for activation (2,3).
Currently, it remains unclear how inflammasome activation is orchestrated across cell types during an infection and whether each cell type has the same ability to activate the inflammasome and release inflammatory cargo.To investigate this question, we employed an in vivo mouse model of Toxoplasma gondii infection, in which the activation of the inflammasome has been reported to result in the release of various immunostimulatory molecules.
T. gondii is an obligate intracellular parasite that can infect nearly all nucleated cells.The parasite replicates throughout the body of the host during the acute phase of infection and then resides in the brain for the duration of the host's life as a chronic infection.Host survival depends on the adaptive immune system's ability to produce interferon gamma (IFN-γ) (4-7).IFN-γ signaling stimulates the expression and production of proteins that facilitate intracellular pathogen-killing mechanisms, enabling the control of the parasite (8)(9)(10).The dependence on IFN-g underscores the vital role of innate immune sensing, which initiates production of cytokines like IL-12 and IL-18 that bridge the innate the adaptive immune system.In mice, TLR11/12 molecules on dendritic cells recognize T. gondii profilin resulting in MyD88-dependent IL-12 release (11)(12)(13)(14).IL-12 acts on natural killer (NK) and T lymphocyte cells to promote an IFN-γ response (15)(16)(17).Of note, humans lack functional TLR11/12 and can still mount an effective immune response to T. gondii, suggesting that other innate sensors recognize and respond to the parasite.One such sensor is the inflammasome, which has been suggested to promote protective immunity during acute infection in humans, rats, and mice (18)(19)(20)(21)(22).At present, in vitro studies and survival studies have been performed but which cells utilize caspase-1 at various stages of T. gondii infection and the immunological consequences have not been defined.
The inflammasome mediates the release of cytoplasmic cytokines (leaderless cytokines) and premade immunostimulatory molecules (alarmins).In vitro and in vivo studies have shown that T. gondii infection promotes the release of leaderless cytokines IL-1α, IL-1β, IL-33, and IL-18 and the alarmin s100a11 (18,19,21,(23)(24)(25)(26)(27)(28).A recent study by López-Yglesias, et.al demonstrated that in the absence of TLR11, inflammasome effector proteins caspase-1/11 promote CD4 + T cell IFN-γ production (20).Another study observed that enhancing IL-18's ability to bind cell surface receptors can promote stronger CD4 + T cell and NK cell IFN-γ production during T. gondii infection (29).Additionally, it was reported that during acute infection IL-1 signaling onto its cognate receptor IL-1R is required for neutrophil recruitment to the site of infection, but this signal was dispensable for IFN-g responses (24).Furthermore, during acute infection, the alarmin s100a11 promotes a Ccl2-mediated monocyte recruitment to the site of infection.Together, these studies indicate that caspase-1/11 may play a role in multiple facets of the immune response to T.
gondii.However, it remains uncertain whether caspase-1 specifically is necessary and whether caspase-1 in distinct cell types performs specific roles in inflammasome activity or if a single population is responsible for the release of multiple leaderless cytokines and alarmins.
To investigate caspase-1-mediated immunity against T. gondii, we used whole-body and cell-typespecific knockout mice.Using this approach, we report that caspase-1 is required for optimal parasite control, promoting CD4 + T cell IFN-γ production and enhancing neutrophil and monocyte recruitment to the initial site of infection.In particular, we find that Cx3cr1-positive cells use caspase-1 to release IL-18, which reinforces IFN-γ production.On the other hand, monocyte and neutrophil responses were intact in these mice, which suggests that caspase-1 activity in additional cell types regulates these innate responses.Our studies demonstrate that in the context of in vivo T. gondii infection caspase-1 is utilized in distinct cell populations to carry out non-overlapping and specific downstream effects of caspase-1-mediated immunity to T. gondii.

Caspase-1 mediates aspects of innate and adaptive immunity to T. gondii.
The initial studies examining the role of inflammasomes in T. gondii infection were performed in double knockout mice, in which expression of both Casp1 and -11 were ablated and immunity against the parasite was impaired (18)(19)(20).We hypothesized that the caspase-1 canonical inflammasome pathway would be sufficient to drive multiple aspects of protective immunity towards T. gondii.Therefore, we infected Casp1 single knockout mice with 10 cysts of Me49 type II strain parasite by intraperitoneal (i.p.) infection (30).Then, we performed peritoneal lavage at eight days post-infection (8 dpi) and found that the parasite burden was 3.6-fold greater in Casp1 deficient mice compared to WT controls (Fig 1a).Additionally, during chronic infection at six weeks post-infection (6 wpi), we found a 1.4-fold increase in parasite burden in the brains of Casp1  (19,20,31).
As IFN-γ is a major regulator of immunity to T. gondii, we then aimed to identify cell populations where IFN-γ production was impacted by Casp1 deficiency.We harvested spleen and peritoneal exudate cells at 8 days post-infection and used spectral flow cytometry to identify IFN-γ-producing cell populations.We found fewer CD4 + T cells that produced IFN-γ within the spleen in Casp1 Beyond a defect in IFN-g production, we observed a dramatic decrease in the number of neutrophils (CD11b + Ly6G + ) and monocytes (CD11b + Ly6G -Ly6C + ) recruited to the peritoneum in Casp1 KO mice compared to WT mice (Fig 1h-l).Corresponding to the decreased monocyte infiltration, in the peritoneum we found decreased levels of the mRNA for the monocyte chemoattractant, Ccl2, in the peritoneum of Casp1 KO mice compared to WT (Fig 1j).Similarly, mRNA levels of Cxcl1 and Cxcl2, major chemokines for neutrophil attraction, were decreased in whole-body Casp1 KO peritoneal lavage fluid (Fig 1m).Together these data demonstrate that Casp1 deficiency leads to innate and adaptive immune defects that are associated with an inability to fully control parasite levels during acute T. gondii infection.

IL-18 promotes CD4 + T cell IFN-γ production but is dispensable for myeloid cell recruitment to the initial site of infection.
We hypothesized that the decreased IL-18 serum levels in whole-body Casp1 knockout mice impaired CD4 + T cell IFN-γ production.Thus, we infected Il18 KO and WT control mice to gain insight on which immune responses downstream of caspase-1 activation were dependent on IL-18.Mice were infected with 10 cysts Me49 followed by analysis of peritoneal exudate cells and spleens at eight days post-infection.Similar to Casp1 deficient mice, Il18 deficient mice displayed an increased parasite burden in the peritoneal cavity and decreased serum IFN-γ levels (Fig 2a and

IL-1Rap mediates neutrophil recruitment but is dispensable for parasite control at the site of infection.
IL-18 is one of many molecules that can be released downstream of caspase-1.To broadly test if the loss of other leaderless cytokine signaling could recapitulate whole-body capase-1 deficiency during acute T. gondii infection, we utilized IL-1 receptor accessory protein (Il1rap) knockout mice.IL-1RAP is a necessary receptor subunit for IL-1, IL-33, and IL-36 receptor signaling.We infected Il1rap WT and KO mice with 10 cysts Me49 and performed peritoneal lavage at eight days post infection.We observed no difference in parasite burden between Il1rap KO and WT control mice at this site (Fig 3a).Furthermore, we observed no difference in IFN-γ serum levels or splenic CD4 + T cell IFN-γ production (Fig 3b-d).In the peritoneum, Il1rap KO mice had comparable monocyte responses (Fig 3e-g).However, there was a severe impairment in neutrophil recruitment to the site of infection in Il1rap KO mice (Fig 3h-j).These results are in line with a previous report showing that IL-1R signaling is required for neutrophil recruitment during acute infection and is dispensable for IFN-γ responses (24).Taken together, these data suggest that IL-1RAP signaling is critical for neutrophil responses but is dispensable for parasite control in the presence of IFN-γ and monocytes.

Caspase-1 in Cx3cr1-expressing cells is necessary for parasite control and optimal production of IFN-g by CD4 + T cells
To begin to address how caspase-1 activation shapes immunity to T. gondii, we sought to identify which cells type(s) are involved.We aimed to identify the population responsible for caspase-1mediated IFN-γ production and parasite control.To this end, we utilized a single-cell RNA sequencing dataset that examined gene expression in the spleen at day 14 post-infection with T. gondii.We focused on cell types that express IL-18, as the release of this cytokine is significantly decreased in Casp1 deficient mice.At this timepoint, we confirmed that macrophages are a major source of Il18 and Casp1 expression (S3a-S3b Fig) .We hypothesized that caspase-1 activity in macrophages would be necessary for parasite control, optimal cytokine production, and cellular recruitment to the peritoneal cavity.To test this hypothesis, we crossed mice that constitutively express cre recombinase under the Cx3cr1 promoter to a Caspase-1 fl/fl background (32,33).We infected Cx3cr1 cre/+ x Caspase 1 fl/fl and Cx3cr1 +/+ x Caspase 1 fl/fl littermate controls with 10 cysts Me49 and at eight days post infection we harvested tissues.For simplicity, we have abbreviated Cx3cr1 cre/+ x Caspase 1 fl/fl as macrophage (MФ) Casp1 KO, given that macrophages represent a large portion of the Cx3cr1 + population (32,34).In the peritoneum, we saw a ~3-fold increase in parasite burden in the macrophage Casp1 KO mice compared to control mice (Fig 4a), which was associated with a greater cyst burden in the brains of these mice at 6 weeks post-infection (6wpi) (S3c Fig) .At day eight post-infection, we performed ELISAs on the serum and found that macrophage Casp1 KO mice had decreased IFN-γ levels, comparable IL-12 levels, and decreased IL-18 levels compared to WT mice (Fig 4b-d).These data suggest that a Cx3Cr1 + cell population promotes IL-18 release and enhances IFN-γ production.Using spectral cytometry, we found that macrophage Casp1 KO mice had impaired production of IFN-g in splenic CD4 + T cells (Fig 4e g).IFN-γ production by splenic CD8 + T cell was unimpaired, as was IFN-γ production by peritoneal CD4 + and CD8 + T cells (S3b-S3d Fig) .When we examined neutrophil and monocyte recruitment to the peritoneal cavity, both responses were intact and comparable to WT levels (Fig 4h-m).These data show that macrophage Casp1 KO mice exhibited a selective T cell defect are nearly identical in phenotype to the Il18 knockout mice.

Recombinant IL-18 administration rescues CD4 + T cell IFN-γ production and parasite control in macrophage caspase-1 deficient mice.
Macrophage Casp1 KO mice had decreased serum IL-18 levels and phenocopied IL-18 KO mice with both having selective impairment in CD4 + T cell IFN-γ production.Thus, we hypothesized that the decreased IL-18 levels in macrophage Casp1 KO mice leads to decreased CD4 + T cell IFN-γ production and parasite control.To test this hypothesis, we performed a rescue experiment, where we administered recombinant IL-18 (rIL-18) at 10μg/kg or PBS to macrophage Casp1 deficient mice at 1 hour post-infection and on days 1, 3, 5, and 7 post-infection (Fig 5a).We confirmed that the rIL-18 treatment restored IL-18 serum levels (Fig 5b).As expected, parasite burden was greater in macrophage Casp1 KO mice that received PBS treatment compared to WT controls.However, in macrophage Casp1 KO mice that received rIL-18, parasite burden was comparable to levels seen in WT mice (Fig 5c).Furthermore, we found that rIL- Together, these data demonstrate that administering rIL-18 in macrophage Casp1 KO mice was sufficient to restore IFN-γ production and ultimately recuperate parasitic control.We hypothesized that the recovery of IFN-γ serum levels in mice that received rIL-18 administration would coincide with a recovery in frequency and number of splenic CD4 + T cells making IFN-γ.Indeed, when flow cytometry was performed on splenic cells at 8 dpi, we found that rIL-18 was able to restore IFN-γ production to splenic CD4 + T cells in macrophage Casp1 KO mice (Fig 5f-h).Thus, administration of rIL-18 was sufficient to rescue CD4 + T cell IFN-γ production and parasite control in mice lacking caspase-1 production in Cx3cr1 + expressing cells.Taken together, these results highlight the importance of caspase-1 in Cx3cr1-positive populations to promote IL-18 release.
These studies demonstrate the varied contributions of caspase-1-mediated pathways during an infection.One arm of the caspase-1 response mediates IL-18 release to impact T cell cytokine production and pathogen control, while other arms stimulate signaling (including through IL-1RAP), to mediate cellular recruitment to the site of infection.

DISCUSSION
Prior studies on caspase-1/11 during T. gondii infection have revealed the capacity of inflammasomes to release various molecules in vivo and in vitro (18-20, 23, 35).However, prior to the current study, the necessity of caspase-1 rather than caspase1/11 and the specific cell types utilizing caspase-1 and the immunological responses triggered by caspase-1 activity in vivo were unknown.Traditionally, studies on caspase-1 have employed in vitro cultures utilizing a single cell type.The development of caspase-1 fl/fl mice has allowed for a more detailed exploration of caspase-1 activity in vivo (33).This study presents evidence indicating that distinct cell populations use caspase-1 to support different facets of immunity to T. gondii.Specifically, Cx3cr1-positive populations utilize caspase-1 to promote IL-18 release, which facilitates CD4 + T cell IFN-γ production.In contrast, another population(s) leverages caspase-1 for neutrophil recruitment through IL-1RAP signaling and monocyte recruitment via induction of the chemokine CCL2.
We identified a caspase-1/IL-1RAP signaling axis as a necessary component of neutrophil recruitment to the site of infection.The role of neutrophils during acute T. gondii has been an active area of investigation.A previous report using an anti-Gr-1 antibody to deplete neutrophils suggests that neutrophils are critical for parasite control (36).Anti-Gr-1 is not specific to neutrophils and results in the depletion of inflammatory monocytes, making it difficult to interpret the importance of neutrophils during acute T. gondii infection.Interestingly, a recent in vitro study showed that T. gondii-induced neutrophil extracellular trap (NET) formation promoted the migration and cytokine response of T cells, suggesting that neutrophils can promote a more robust adaptive immune response (37).A third study using an antibody targeting Ly6G to obtain a more selective depletion of neutrophils found no role for neutrophils in parasite control or IFN-γ levels in orally infected mice (38).Similarly, our study using Il1rap KO mice which only had a defect in neutrophil recruitment to the site of infection suggests that neutrophils are not necessary for T. gondii control or IFN-γ production.Interestingly, both IL-33 signaling (ST2) and IL-1 signaling (IL-1R1) utilize IL-1RAP and have been associated with neutrophil recruitment during acute T. gondii infection (24,39).In both of these studies the knockout of each respective receptor brought neutrophil levels drastically down.Thus, although neutrophil recruitment is regulated through caspase-1 and IL-1RAP signaling, this specific immunological response to T. gondii appears to be dispensable for parasite control and is in agreement with studies that used anti-Ly6G antibodies to deplete neutrophils.Whether caspase-1 mediates the release of IL-33, IL-1β, and/or IL-1α to promote neutrophil recruitment remains unresolved.Caspase-1 cleavage of IL-33 is suggested to inactive the cytokine (40) but the mechanism of IL-33 release is still under investigation (41).Recent studies have identified that IL-33 is released from cells via gasdermin D pore formation, but the protease activating gasdermin D varied with the stimulus used to induce IL-33 release (42,43).Thus, whether caspase-1 liberates IL-33 or inactivates IL-33 during T. gondii infection is of interest.
Our study on Il1rap KO mice not only provided insight the role of neutrophils during T. gondii infection but also on monocyte recruitment.As both Il18 KO and Il1rap KO mice had no defect in monocyte recruitment, our data suggests an additional signal is promoting monocyte recruitment to the site of T. gondii infection.Our study aligns with a recent report describing how monocytes are recruited to the site of infection.Specifically, Safronova, et al. recently described that the alarmin S100a11 mediates Ccl2 expression and monocyte recruitment to the peritoneal cavity following infection.In accordance, they found that S100a11 release was caspase-1/11-dependent, both in vitro and in vivo (23).Together with our study, this suggests that a population (s) other than macrophages is driving caspase-1/S100a11-/CCL2-dependent monocyte recruitment.
Our study is also in accordance with a recent study from Clark et.al., which describes that IL-18 and its regulation impacts the ability of CD4 + T cells to make IFN-γ (29).Although, initially we were surprised to see that the NK cell IFN-γ response is intact in Casp1 and Il18 KO mice, López-Yglesias, et.al. also showed that Casp1/11 deficiency had no impact on NK cell IFN-γ responses.One likely explanation is that the timing of IL-18 interaction with NK cells is critical.It has been observed that if an NK cell interacts with IL-12 before interacting with IL-18, IL-18 will not impact NK cell IFN-γ production (44).During T. gondii infection IL-12 levels are detected and increase before IL-18 levels (45-47), thus NK cells may encounter IL-12 well before IL-18, explaining why IL-18 has minimal impact on NK cell activation.We were also surprised by the specificity of the IFN-g defects in splenic CD4 + T cells and not CD8 + T cells in Casp1, Il18, and macrophage Casp1 knockouts.These results can be explained by the preferential upregulation of IL-18R on CD4 + T cells at day 8 post-infection.Interestingly, the report by Clark et.al. demonstrates that CD8 + T cells upregulate IL-18R expression at day 10 post-infection (29).Thus, the expression of IL-18R on CD8 + T cells is tightly regulated by an unknown cue.Similarly, we found that NK cell/ILC1 cell population expression of IL-18R is absent at baseline and present at day eight post-infection, whereas, in accordance with Clark et.al., the NK T cell population expressed IL-18R at baseline and at day eight (29).Interestingly, our study and the study from Clark et.al. showed that exogenous addition of IL-18 did not impact NK cell IFN-γ production, but Clark et.al. showed that when a recombinant IL-18 resistant to inhibition by IL-18 binding protein was exogenously added, NK cell responses and IFN-γ production were enhanced.These data suggest that the response of NK cells to IL-18 is highly regulated.Overall, these data suggest that IL-18 release and IL-18R expression are exquisitely controlled and ultimately influence the quality of CD4 + T cell production of IFN-g.
While the current study has identified the importance of caspase-1 activity in specific cell populations, the signal(s) that leads to inflammasome activation remain unclear.Additionally, as multiple inflammasome sensors have shown to be triggered by T. gondii, it is possible that in the Cx3cr1-positive and other populations, caspase-1 is activated by distinct mechanisms and sensors.
In vitro, both direct T. gondii infection and extracellular signaling cascades have been proposed to initiate the inflammasome (18,27,48,49).Cell-intrinsic signals and cellular stress are also possible modes of inflammasome activation (50,51).Although, during in vivo T. gondii infection, it is unclear whether direct invasion of the parasite and/or an infection-associated trigger is necessary for caspase-1 activation.A recent study by Wang, et. al., identified that in bone marrow derived macrophages (BMDMs) from the Lewis rat, NLRP1-dependent pyroptosis is mediated by direct infection and three parasite dense granule proteins GRA35, 42, and 43 (27).In the human monocyte cell line (THP-1) cell cultures infected with T. gondii, the AIM2 inflammasome sensor has been shown to be activated by T. gondii DNA that has been liberated by guanylate binding protein (GBP) activity (48,52).A third study using primary mouse peritoneal macrophages and BMDMs identified that NLRP3 was activated by extracellular ATP signaling released from parasite infected cells (49).Our data along with these in vitro experiments raise the question of whether certain cell types can activate multiple inflammasome sensors and release multiple leaderless cytokines or DAMPs.In the single cell data set we analyzed, the splenic macrophage population containing IL-18 expressed AIM2 more than other inflammasome sensors (29).We did not observe prominent expression of NLRP3 within the splenic macrophage population which is consistent with work from López-Yglesias et.al., which showed that NLRP3 does not impact CD4 + T cell IFN-γ production during acute T. gondii infection (20).NLRP1 which has been shown to confer resistance to T. gondii infection in humans and rats (18,21,35) and has been reported to modulate IL-18 release in mice (19), was expressed in only a small percentage of splenic IL-18expressing cells.Ultimately, whether one sensor or multiple sensors contribute to the distinct caspase-1 activities remains an open question.
In addition, the responses downstream of inflammasome activity are influenced by cellular expression of leaderless cytokines, leaderless cytokine receptors, and the chemokines a receptive cell can produce.Furthermore, the spatial location of these interactions likely influences the effects of inflammasome activity.For instance, IL-1α and IL-1β are regulated by a soluble receptor antagonist (IL-1R antagonist) which competes with surface receptors.Thus, efficient IL-1 signaling may require local proximity of a IL-1R1-expressing cell (53).Similarly, IL-18 is regulated by IL-18BP, making the proximity of IL-18 release near responding CD4 + T cells critical for optimal IFN-γ production.To parse out this complex and specific interplay, high-dimensional spatially-resolved data sets will be of value.In this study, we were able to use a previously published single-cell RNA sequencing dataset to aid in the identification of a putative cell population capable of activating caspase-1 and releasing IL-18.The use of spatial transcriptomics and its ability to identify cells harboring leaderless cytokines and identify proximal cells able to respond to the cytokine will be advantageous for future studies on the orchestration of local inflammasome responses.The current study also demonstrates that distinct cell types use caspase-1 to impact innate and adaptive immune responses, which may provide an advantage to the host.
The separation of the potent effects of caspase-1 activity across multiple cell types with differential inflammasome activation capacity likely acts as a checkpoint to prevent excess inflammation.
Having a layered regulation of inflammasome activation in vivo allows for a broad range of activators to elicit specific and non-redundant responses.
As our study focused on the role of caspase-1 during acute T. gondii infection, it is unclear if a similar paradigm of cell-type specificity occurs in the brain during chronic T. gondii infection.As the brain contains long-lived cells which do not regenerate, the control of inflammation in this tissue is critical.A layered regulation of caspase-1 activity in the CNS would be advantageous for controlling T. gondii without promoting excess deleterious inflammation.A recent study by our group (24) identified that microglia, a Cx3cr1-positive cell population, releases IL-1α to promote myeloid cell recruitment into the infected brain and control parasite.This contrasts with acute infection where a population(s) other than Cx3cr1-positive cells contributes to myeloid recruitment.This highlights a significant difference in caspase-1 biology in the infected CNS and raises the important question of whether the CNS contains multiple cell types capable of activating caspase-1 to carry out unique aspects of immunity.Given that Casp1 and Casp1/11 deficient mice have higher parasite burdens in the CNS, it suggests that inflammasome activity is necessary in the brain and likely regulates local immune responses.
For experiments, tissue cysts were collected from chronically infected (>4 weeks) CBA/J mice.7-10-week-old age and sex-matched mice were injected intraperitoneally with 10 cysts of Me49 in 200 µL of 1X PBS.Sham-infected mice were injected with an equal volume of 1X PBS.For rIL-18 experiments, 10 μg/kg or PBS of equivalent volume was given to mice on day 0, 1, 3, 5, and 7 post-infection with T. gondii.Mice used for endpoint studies were euthanized if they showed weight loss greater than 20% of their pre-infection bodyweight.All procedures involving animal care and use were approved by and conducted in accordance with the University of Virginia's Institutional Animal Care and Use Committee (IACUC) under protocol number 3968.

T. gondii qPCR
Parasite genomic DNA was isolated from mouse peritoneal exudate cells and whole brain using the Isolate II Genomic DNA Kit (Bioline, BIO-52067).Prior to isolation, brain was first homogenized in 1X PBS using the Omni TH tissue homogenizer (Omni International).Amplification of T. gondii 529 bp repeat region using the SensiFAST Probe No-ROX Kit (Bioline, BIO-86005) and CFX384 Real-Time System (Bio-Rad) was performed as previously described (57) Tissue DNA (500 ng or 1µg) was loaded into each reaction.T. gondii isolated from human foreskin fibroblasts was used to make a serial standard curve from 3 to 300,000 genome copies and determine the number of T. gondii genomes per µg of tissue DNA.
KO mice relative to control mice (S1a Fig).Surprisingly, Casp1 and Casp1/11 KO mice had similar long-term survival post-infection compared to WT mice (S1b Fig), but consistently had higher parasite burdens.To test if the increased parasite burden at day 8 was due to an immunological defect, we performed ELISAs on the serum of WT and Casp1 KO mice.We found that Casp1 KO mice had a 25% reduction in IFN-γ levels compared to WT controls (Fig 1b) but had comparable IL-12 serum levels (Fig 1c).On the other hand, we found that IL-18 serum levels in Casp1 KO mice were nearly half of what was measured in WT controls (Fig 1d).We were unable to detect IL-1α or IL-1β in the serum at this timepoint, consistent with prior reports KO mice in comparison to WT controls (Fig 1d-f).Casp1 was dispensable for splenic CD8 + T cells, NK cells, and ICL1/NK T cells IFN-γ production (S1c-S1e Fig).Additionally, in the peritoneum 8 dpi, no consistent defect was seen in IFN-γ production by CD4 + and CD8 + T cells or by NK/ ILC1 and NK T cells (S1f-S1i Fig).Together these data highlight a defect in IFN-g production specific to splenic CD4 + T cells in mice lacking Casp1.

Figure 3 .
Figure 3. IL-1RAP signaling mediates neutrophil recruitment to the sight of infection but is

Figure 4 .
Figure 4. Caspase-1 in Cx3cr1-expressing cells is necessary for parasite control and optimal